The blood coagulation cascade is divided into three distinct segments: the intrinsic, extrinsic, and common pathways (Schenone et al., Curr Opin Hematol. 2004; 11:272-7). The cascade involves a series of serine protease enzymes (zymogens) and protein cofactors. When required, an inactive zymogen precursor is converted into the active form, which consequently converts the next enzyme in the cascade.
The intrinsic pathway requires the clotting factors VIII, IX, X, XI, and XII. Initiation of the intrinsic pathway occurs when prekallikrein, high-molecular-weight kininogen, factor XI (FXI) and factor XII (FXII) are exposed to a negatively charged surface. Also required are calcium ions and phospholipids secreted from platelets.
The extrinsic pathway is initiated when the vascular lumen of blood vessels is damaged. The membrane glycoprotein tissue factor is exposed and then binds to circulating factor VII (FVII) and to small preexisting amounts of its activated form FVIIa. This binding facilitates full conversion of FVII to FVIIa and subsequently, in the presence of calcium and phospholipids, the conversion of factor IX (FIX) to factor IXa (FIXa) and factor X (FX) to factor Xa (FXa). The association of FVIIa with tissue factor enhances the proteolytic activity by bringing the binding sites of FVII for the substrate (FX and FIX) into closer proximity and by inducing a conformational change, which enhances the enzymatic activity of FVIIa. The rate of FX activation by the extrinsic pathway is approximately 50 times slower than the rate achieved by the (intrinsic) pathway of FIXa, FVIIIa, phospholipid, and calcium ions.
The activation of FX is the common point of the two pathways. Along with phospholipid and calcium, factors Va (FVa) and Xa convert prothrombin to thrombin (prothrombinase complex), which then cleaves fibrinogen to form fibrin monomers. The monomers polymerize to form fibrin strands. Factor XIIIa (FXIIIa) covalently bonds these strands to one another to form a rigid mesh.
Conversion of FVII to FVIIa is also catalyzed by a number of proteases, including thrombin, FIXa, FXa, factor XIa (FXIa), and factor XIIa (FXIIa). For inhibition of the early phase of the cascade, tissue factor pathway inhibitor targets FVIIa/tissue factor/FXa product complex.
FVII (also known as stable factor or proconvertin) is a vitamin K-dependent serine protease glycoprotein with a pivotal role in hemostasis and coagulation (Eigenbrot, Curr Protein Pept Sci. 2002; 3:287-99).
FVII is synthesized in the liver and secreted as a single-chain glycoprotein of 48 kD. FVIIa shares with all vitamin K-dependent serine protease glycoproteins a similar protein domain structure consisting of an amino-terminal gamma-carboxyglutamic acid (Gla) domain with 9-12 residues responsible for the interaction of the protein with lipid membranes, a carboxy-terminal serine protease domain (catalytic domain), and two epidermal growth factor-like domains containing a calcium ion binding site that mediates interaction with tissue factor.
Gamma-glutamyl carboxylase catalyzes carboxylation of Gla residues in the amino-terminal portion of the molecule. The carboxylase is dependent on a reduced form of vitamin K for its action, which is oxidized to the epoxide form. Vitamin K epoxide reductase is required to convert the epoxide form of vitamin K back to the reduced form.
The major proportion of FVII circulates in plasma in zymogen form, and activation of this form results in cleavage of the peptide bond between arginine 152 and isoleucine 153. The resulting activated FVIIa consists of a NH2-derived light chain (20 kD) and a COOH terminal-derived heavy chain (30 kD) linked via a single disulfide bond (Cys 135 to Cys 262). The light chain contains the membrane-binding Gla domain, while the heavy chain contains the catalytic domain.
The plasma concentration of FVII determined by genetic and environmental factors is about 0.5 mg/mL (Pinotti et al., Blood. 2000; 95:3423-8). Different FVII genotypes can result in several-fold differences in mean FVII levels. Plasma FVII levels are elevated during pregnancy in healthy females and also increase with age and are higher in females and in persons with hypertriglyceridemia. FVII has the shortest half-life of all procoagulant factors (3-6 h). The mean plasma concentration of FVIIa is 3.6 ng/mL in healthy individuals and the circulating half-life of FVIIa is relatively long (2.5 h) compared with other coagulation factors.
Hereditary FVII deficiency is a rare autosomal recessive bleeding disorder with a prevalence estimated to be 1 case per 500,000 persons in the general population (Acharya et al., J Thromb Haemost. 2004; 2248-56). Acquired FVII deficiency from inhibitors is also very rare. Cases have also been reported with the deficiency occurring in association with drugs such as cephalosporins, penicillins, and oral anticoagulants. Furthermore, acquired FVII deficiency has been reported to occur spontaneously or with other conditions, such as myeloma, sepsis, aplastic anemia, with interleukin-2 and antithymocyte globulin therapy.
Replacement therapy is the mainstay of treatment for patients with FVII deficiency (Mariani et al., Semin Hematol. 2006; 43(Suppl 1):S42-7). This has traditionally been achieved using fresh frozen plasma (FFP), prothrombin complex concentrates (PCCs), or plasma-derived FVII concentrates. However, recombinant FVIIa (rFVIIa) is now widely used for therapy in these patients.
RFVIIa has also been developed for treatment of bleedings in hemophilia A and B patients with inhibitors, and has been found to induce hemostasis even during major surgery such as major orthopedic surgery (Hedner, J Biotechnol. 2006; 124:747-57). RFVIIa is being produced in BHK cell cultures and has been shown to be very similar to plasma-derived FVIIa. The use of rFVIIa in hemophilia treatment is based on the low affinity binding of FVIIa to the surface of thrombin activated platelets. By the administration of pharmacological doses of exogenous rFVIIa the thrombin generation on the platelet surface at the site of injury is enhanced independently of the presence of FVIII/FIX. As a result of the increased and rapid thrombin formation, a tight fibrin hemostatic plug is being formed.
Although originally developed for the treatment of FVII deficiency and inhibitor-complicated hemophilia A and B, novel indications for rFVIIa (based on case reports and smaller clinical trials) include use in patients with liver disease, thrombocytopenia, or qualitative platelet dysfunction and in patients with no coagulation disorders who are bleeding as a result of extensive surgery or major trauma.
Therapeutic polypeptide drugs such as blood coagulation protein including FVIIa are rapidly degraded by proteolytic enzymes and neutralized by antibodies. This reduces their half-life and circulation time, thereby limiting their therapeutic effectiveness. Relatively high doses and frequent administration are necessary to reach and sustain the desired therapeutic or prophylactic effect of FVIIa. As a consequence adequate dose regulation is difficult to obtain and the need of frequent intravenous administrations imposes restrictions on the patient's way of living. Thus an improved FVIIa molecule with a longer circulation half-life would decrease the number of necessary administrations.
In principal, there are four general options for half-life extension of proteins in the blood circulation:                Direct chemical or enzymatic modification        Use of carrier molecules to protect the proteins in the circulation        Construction of mutants to extent half-life        Modification of the degradation pathway.        
The present invention teaches an improvement of blood coagulation proteins, especially the FVIIa molecule by chemical modification. For chemical modification of therapeutic polypeptides several approaches have been used in the past.
PEGylation of polypeptide drugs protects them and improves their pharmacodynamic and pharmacokinetic profiles (Harris and Chess, Nat Rev Drug Discov. 2003; 2:214-21). The PEGylation process attaches repeating units of polyethylene glycol (PEG) to a polypeptide drug. PEG molecules have a large hydrodynamic volume (5-10 times the size of globular proteins), are highly water soluble and hydrated, very mobile, non-toxic, non-immunogenic and rapidly cleared from the body. PEGylation of molecules can lead to increased resistance of drugs to enzymatic degradation, increased half-life in vivo, reduced dosing frequency, decreased immunogenicity, increased physical and thermal stability, increased solubility, increased liquid stability, and reduced aggregation. The first PEGylated drugs were approved by the FDA in the early 1990s. In the meantime the FDA approved several PEGylated drugs for oral, injectable, and topical administration.
GlycoPEGylation™ technology includes methods that provide a peptide conjugate between a PEG polymer and a peptide, with the PEG covalently attached to the peptide via an intact glycosyl-linking group.
Liposomes have been used to encapsulate a variety of molecules such as DNA, anti-sense RNA, antibiotics, anti-cancer, and anti-fungal drugs, inhibitors/activators, antibodies (immunoliposomes), and antigens (for vaccines).
Phospholipids can be also conjugated to PEGs (PEG-liposome) for example via an amide linkage, carboxy-PEG and purified soy phosphatidylethanolamine (PE), esters and carbamate derivatives, the carbamate derivative being the most widely used today (U.S. Pat. No. 6,593,294). The molecular weights of the most commonly used PEG's are 2,000 and 5,000, but PEG's ranging from 600 to 12,000 are also used.
Acidic monosaccharide-substituted proteins were first disclosed in U.S. Pat. No. 3,847,890. In this patent acidic monosaccharides, i.e. n-acetylneuraminic acid and gluconate were substituted onto α-amino or ε-amino groups of insulin, human growth hormone or albumin to reduce the antigenicity of the polypeptides.
Polysialic acid (PSA), also referred as colominic acid (CA), is a naturally occurring polysaccharide. It is a homopolymer of N-acetylneuraminic acid with α(2→8) ketosidic linkage and contains vicinal diol groups at its non-reducing end. It is negatively charged and a natural constituent of the human body. It can easily be produced from bacteria in large quantities and with pre-determined physical characteristics (U.S. Pat. No. 5,846,951). Being chemically and immunologically identical to polysialic acid in the human body, bacterial polysialic acid is non-immunogenic, even when coupled to proteins. Unlike other polymers (eg. PEG), polysialic acid is biodegradable. Covalent coupling of colominic acid to catalase and asparaginase led to an increase of enzyme stability in the presence of proteolytic enzymes or blood plasma. Comparative studies in vivo with polysialylated and unmodified asparaginase revealed that polysialylation increased the half-life of the enzyme (Fernandes and Gregoriadis, Int J Pharm. 2001; 217:215-24)
However, to date no therapeutic compounds consisting of a polypeptide conjugated to an acidic monosaccharide as described in U.S. Pat. No. 3,847,890 are commercially available. In contrast, U.S. Pat. No. 5,846,951 teaches that the polysaccharide portion of the compound should have at least 5, and in other embodiments at least 20 or 50 sialic acid residues in the polymer chain. Because the polysaccharides are usually produced in bacteria carrying the inherent risk of copurifying endotoxins, the purification of long sialic acid polymer chains may raise the probability of increased endotoxin content. Short PSA molecules with a 1-4 sialic acid units can also be synthetically prepared (Kang et al., Chem Commun. 2000; 227-8; Ress and Linhardt, Current Organic Synthesis. 2004; 1:31-46), thus minimizing the risk of high endotoxin levels.
WO 98/32466A1 suggests that FVII, among many other proteins, may be PEGylated but does not contain any working examples supporting the disclosure.
WO 01/58935A3 teaches conjugates comprising at least one non-polypeptide moiety covalently attached to a polypeptide, wherein the amino acid sequence of the polypeptide differs from that of wild-type FVII or FVIIa in that at least one amino acid residue comprising an attachment group for said non-polypeptide moiety has been introduced or removed. For the non-polypeptide moieties especially PEG was suggested.
US20050113565A1 discloses a FVII polypeptide or FVII-related polypeptide, wherein the polypeptide comprises one or more asparagine-linked and/or serine-linked oligosaccharide chains, and wherein at least one of said oligosaccharide groups is covalently attached to at least one polymeric group (PEG, “glycoPEGylation”).
Thus, there remains a need in the art for compositions and methods that provide clotting protein preparations comprising improved plasma derived or rFVII, modified FVII, or FVII-related polypeptide.